This invention relates to a rapid, high throughput method for simultaneously measuring biologically significant physicochemical characteristics for multiple compounds. More particularly, this invention is directed to the measurement and use of binding affinities of multiple compounds with a surface under controlled conditions to calculate biologically significant physicochemical values. In one embodiment, the present invention provides a new method for the determination of a most important biological parameter, membrane interfacial surfacepKa, the knowledge of which significantly facilitates understanding of drug-membrane interactions and thereby speeds the drug discovery process.
Many potential new drug candidates are created every year, and both pharmaceutical and biotechnology industries have embraced the challenge in recent years of developing faster and more efficient ways to screen pharmaceutical compounds in order to rapidly identify xe2x80x9chitsxe2x80x9d and develop them into promising lead drug candidates. This has created the need for high-throughput analytical approaches to characterize the synthesized compounds and has prompted the development of chromatographic systems specifically designed for the automated high-throughput identification, purity assessment, purification or determination of the physicochemical properties of the compounds in combinatorial libraries.
With the advent of combinatorial chemistry and the need to develop assays for the large numbers of compounds being made available using that technology, many researchers have focused their efforts on developing in vitro tests/assays that provide biologically significant compound information. Much work has been directed to correlation of certain physicochemical properties with biological activity, both in the search for new therapeutic agents and in the understanding of compound toxicity both from medicinal and environmental perspectives.
The rise of combinatorial chemistry and other drug discovery technologies has vastly increased the number of new compounds to be evaluated as potential drug candidates. Accordingly, new high-throughput strategies are required to evaluate compound properties beyond potency and selectivity. A major focus in the pharmaceutical industry is to develop new drugs with good oral bioavailability. Bioavailability represents both the quantity of the drug administered reaching the blood circulation, and the rate of this phenomenon. It has been generally considered that the bioavailability of an orally administered drug is mostly determined by its physicochemical properties; e.g., its molecular weight, pKa, lipophilicity, solubility. One of the factors influencing oral bioavailability is the gastrointestinal pH, as it influences the ionization of the compounds. Most drugs are either weak bases or weak acids, and normally only the non-ionized fraction (i.e., the most lipophilic form) crosses biological membranes, except when transport carriers are involved. It becomes evident that knowledge of the pKa of potential drug candidates may give insight as to how good an oral bioavailability they will exhibit. In particular, having access to the dissociation state of said drug candidates at the membrane interface (surfacepKa) would be helpful, as it would give an indication on their potential oral bioavailability and thus their pharmaceutical value. Thus, one physicochemical property of recognized significance to evaluation of a compound""s biological activity, whether it be therapeutic efficacy or toxicity, is its dissociation constant, more significantly, the dissociation constant exhibited by the compound at a membrane interface.
For an ionizable compound, the degree of dissociation (or protonation) in solution is usually quantified by the pKa value of the acidic form. Thus, in the following acid-base equilibrium equation.
HA⇄Axe2x88x92+H+
the dissociation constant Ka is expressed as:       K    a    =                    [                  H          +                ]            ⁡              [                  A          -                ]                    [      HA      ]      
The pKa of a molecule, defined as xe2x88x92log Ka, is indicative of its degree of ionization, or of its acidic strength in solution. To be able to predict the extent to which a particular compound will ionize at a given pH (for example at physiological pH) is of great importance because it will give insight as to how well the substance in question will be able to participate in physical, chemical and biological reactions. A prominent example from medicinal chemistry is the ability of drugs to pass through biological membranes as well as their potential to interact with intracellular receptors, both of which are affected by the readiness of the drug to undergo protonation or deprotonation. Many biological processes involve reactions at the surface of, or within, cell membranes. Explanation of these functions in structural terms requires a physicochemical understanding of the various interactions taking place. In many cases the processes concern small amphiphilic molecules with pKa values close to physiological pH, and therefore both charged and uncharged forms may interact with the membrane. At the membrane surface, each form partitions into the membrane to a different extent, depending on the respective membrane affinities (FIG. 2). The membrane-bound solute species are in turn involved in yet another acid/base equilibrium, with a dissociation constant surfaceKa distinct from the above mentioned bulkKa. The surfacepKa (interfacial/surface pKa) is frequently biologically relevant, as it is the true indicator of the ionization state of the solute at the membrane interface, and therefore provides fundamental information of the solute/membrane interactions.
Several studies have demonstrated the key role that solute ionization plays in solute/membrane interactions. Biological membranes are made up of a lipid bilayer, which is a complex mixture of lipids and proteins held together mainly by non-covalent interactions. In such mixtures, phase separation leading to domain formation is possible, where the lipids are not distributed uniformly but rather in xe2x80x9cclustersxe2x80x9d. The domains"" surfaces differ in physicochemical properties, and a particular chemical (drug) is expected to have different membrane binding and partitioning behaviors (dictated in part by its surfacepKa) with the membrane domains. This may contribute to accumulation and co-localization of receptors and drugs in the same (small and specialized) membrane region. It has been shown that solute ionization can determine the depth of membrane penetration and location of solutes in membranes, as well as affect the membrane physiology. The local anesthetic tetracaine hydrochloride (TTC) is a good example, because at pH 5.5 the positively charged TTC resides near the phospholipid head group region, whereas at pH 9.0 the uncharged TTC penetrates more deeply into the hydrocarbon region of the membrane. This membrane penetration behavior significantly affects the expansion of the membrane, which has been proposed as one aspect of the mechanism of action of anesthetics. This study not only demonstrates that membrane physiology is affected by charged versus neutral molecules, but also that the activity of the compound, and the depth of its membrane penetration depend on its ionization state at the membrane surface and not in the bulk solution.
In addition to affecting the location of molecules in cell membranes, solute ionization also affects solute movement from the outer monolayer to the inner monolayer of cell membranes: for example, it has been reported that the migration of unionized fatty acids across the phospholipid bilayer is much greater than for their ionized counterparts. The rapid flip-flop of unionized fatty acids has important physiological implications as it enables the rapid entry and removal of fatty acids from cells such as adipocytes, hepatocytes, and heart muscle tissue. Movement between the inner and outer monolayer of cell membranes is necessary for transport not only of fatty acids but also many biologically important compounds. For example it has been reported that the ability of certain CNS compounds to cross the blood-brain barrier is affected by their ionization state. The antiarrhythmic aminoxylidides have a pKa ranging from 4.8 to 8.0. Assuming a constant overall concentration of the drug and a physiological pH of 7.4, the concentrations of the ionized forms of the drug molecules could vary by as much as 2.5 logarithmic units, and 0.7 log unit for the unionized species. That study emphasizes that any changes in pKa can lead to a dramatic difference in concentration of the drug molecules at the membrane surface. This also demonstrates the importance of the interfacial pKa as a useful predictive parameter of a drug""s potential activity, toxicity and ability to transport through the membrane bilayer.
Solute ionization also influences the binding energy between solutes and cell membranes, which is an important parameter associated with the transport of solutes across membranes. Finally, and probably most importantly, the solute""s ionization state can play a critical role in the biological activity of the molecule in question. For example, the activity of protein kinases C (PKC) requires the presence of negatively charged acidic phospholipids. One putative mechanism to inhibit protein kinase C is to neutralize negatively charged phospholipids at the cell membrane. N,N-Dimethyl-D-sphingosine (DMS), a known inhibitor of PKC and a potential antitumor agent, has a membrane bound pKa value of 8.8. As a result of this high pKa, 90% of DMS is protonated at physiological pH which causes inhibition of PKC activity. Other analogs in the sphingosine family also display this charge dependent inhibitory activity. The key point is that the ionized DMS species is believed to be the active chemical species and understanding solute ionization may allow similar conclusions for other compounds. Being able to determine whether the ionized versus unionized species is biologically active may thus be important to drug design.
Similarly, the degree of ionization is an important factor for the toxicity and fate of organic compounds in natural waters, and specific modes of toxic action, such as the uncoupling of oxidative phosphorylation, depend directly on both the lipophilicity and the acidity of the chemical compound. The bioavailability of chemical contaminants in the environment is governed by physicochemical properties and chemical reactivity, which also determine their ability to bioaccumulate and exert certain modes of toxic action in organisms. Knowing the pKa of a particular compound is important because it gives a partial understanding of its partitioning and concentration in the surrounding biological systems and thus gives insights about its ecotoxic potential. The degree of ionization of a chemical can affect its ability to bind to certain soils and sediments, or its interaction with certain living organism cell membranes and thus its toxicity. For example, the aquatic toxicity of several chlorophenols and nitrophenols has been evaluated in different biological systems (bacteria, plant, and fish among others). These phenolic compounds are known to be oxidative uncouplers, and in eukaryotic species, the mechanism of that action is believed to be by destruction of the electrochemical membrane potential by carrying protons into mitochondria, thus depleting the energy needed for ATP formation. Thus pKa is an important parameter capable of predicting the potential environmental toxicity of chemicals. Such knowledge is also valuable from the practical viewpoint of implementing and surveying water quality criteria for instance.
Another aspect also related to toxicity is exemplified by chemicals as toxic gases, synthetic chemicals, cosmetics and topical medicines, which have the ability of entering the body through the skin. These percutaneous transport processes are heavily dependent on the ionization state of the compounds under consideration. The skin permeability of chemicals is associated with their ionization state which affects their potential toxicity.
The classical method for the calculation of bulk pKa is given by the Hammet equation, which is based on the classification of the compound of interest into a parent structure with substituents of known increment values. The determination of pKa""s by this method is straightforward and has been routinely used in structure-activity analyses to evaluate the impact of ionization on bioaccumulation or toxicity. However, application of this classical method is principally restricted to certain types of parent structures and substituents, which may not apply to the compound under investigation. Capillary electrophoresis has also been used for the determination of pKa in solution.
Liquid chromatography-high resolution electrospray mass spectrometry (LC-ESI-MS) has been used for the identification of known and unknown synthetic peptides for peptide sequencing and characterization. The peptides were enzymatically digested using trypsin and the digests analyzed by LC-ESI-MS. The structurally similar peptides resulted in the formation of a number of common tryptic fragments. These tryptic fragments were not always resolved chromatographically for each peptide, but it was possible to select representative CAD (collisionally activated dissociation)-MS spectra from the data and enable amino acid sequencing of the tryptic fragments.
Julian et al. (Julian, R. K. Jr., Higgs, R. E., Gygi, J. D., Hilton, M. D., xe2x80x9cA Method for Quantitatively Differentiating Crude Natural Extracts Using High-Performance Liquid Chromatography-Electrospray Mass Spectrometry.xe2x80x9d Anal. Chem.; 70(15); 3249-3254 (1998)) developed a system that was based on recent advances in chemical analysis and computation to quantitatively characterize and compare complex chemical mixtures. They tested the system on fermentation extracts for drug screening are from 88 uncharacterized fungi. The chemical analysis system is comprised of a pair of HPLC systems to take turns separating the chemical constituents detected by ESI-MS. The system was distinguished from earlier applications of LC-MS in that it was comprised of three components: (a) HPLC separation of the analytes, (b) ESI-MS detection of effluent analytes, and (c) a computational tool to allow effective comparison of the large data sets that are generated by the analysis.
Kassel et al. (L. Zeng, L. Burton, K. Yung, B. Shushan, D. B. Kassel, xe2x80x9cAutomated analytical/preparative high-performance liquid chromatography-mass spectrometry system for the rapid characterization and purification of compound libraries.xe2x80x9d J. Chrom. A., 794:3-13 (1998)) reported an automated parallel analytical/preparative HPLC/MS workstation to increase the analysis speed for characterizing and purifying combinatorial libraries. The system incorporated two columns operated in parallel for both LC/MS analysis and preparative LC/MS purifications. This technique provided a better match for the speed of parallel synthesis. The authors are working on a system with four analytical or preparative columns operated in parallel.
Both spectroscopic and non-spectroscopic methods have been developed to measure surfacepKa. Spectroscopic methods include fluorescence, nuclear magnetic resonance (NMR), and electron spin resonance (ESR). Non-spectroscopic methods include dialysis and surface activity measurement.
For chemically diverse groups of compounds, all of these methods have significant limitations. For instance, fluorescence requires that compounds have a pH-sensitive fluorescent functional group that can be used to quantitate the fraction ionized and unionized at the membrane surface. This is rarely the case. With the exception of fluorescent probes specifically designed to measure the polarity of membrane surfaces, very few solutes possess a fluorescent functional group. ESR methods require spin labeled functional groups that no biologically relevant solute possesses. NMR methods usually have a problem with sensitivity, and consequently milligram quantities of isotope-labeled compounds need to be prepared for each compound. However, the most significant limitation is that most spectroscopic methods have been developed only for Type-1 compounds which exhibit very large membrane partition coefficients such that the solutes are quantitatively membrane-associated at the equilibrium partitioning condition. Except for ESR, spectroscopic methods usually cannot be used for Type-2 compounds that have significant amounts of solute in both the aqueous and membrane environment at the equilibrium partitioning condition. Regarding non-spectroscopic methods like dialysis, which can be used for the latter type of compounds, these methods are experimentally tedious, time consuming, and several measurements are needed to obtain the interfacial pKa. Propagation of error in the experimental measurement also makes the final pKa liposome measurement contain a large amount of uncertainty.
Due to the significant influence of ionization on the location, transport, binding, and activity of molecules, quantitating the ionization state of chemical species by measuring the pKa of membrane associated solutes provides fundamental information that increases our understanding of membrane physiology and solute/membrane interactions. No general method for measuring the pKa of solutes bound to membranes exists. In addition, there is no commercially efficient method that requires minimum amounts of compounds or that allows the high volume determination of bulk pKa""s as is needed today for screening the large number of compounds being made available in combinatorial chemical libraries.
The present invention is directed to a method for determining a physicochemical value for each compound of a set of compounds, such as those that comprise a combinatorial chemical library. The method comprises dissolving the compounds in a plurality of liquid media to form a multiplicity of test solutions of the set of compounds. Each test solution then contacts a surface under a pre-determined set of conditions of temperature and/or pressure. A parameter dependent on the affinity of the surface for each compound in each test solution is measured after contact of the solution with the surface under the pre-determined set of conditions. The measured parameters are then used to calculate the physicochemical value according to a pre-determined algorithm. The present invention provides a method for utilizing chromatographic systems for high-throughput analyses of the physicochemical properties of distinct compounds in a set of compounds. This method allows such determinations to be made in a highly efficient and rapid manner.